We plan to induce, isolate and characterize mutant strains of Physarum polycephalum which are temperature-sensitive (ts) in chromosomal DNA synthesis. Each amoebal mutant carrying a ts dna gene will be used to construct diploid plasmodia homozygous for that mutation. The natural synchrony of the nuclear division cycle of the plasmodium will be exploited in the characterization of the mutants: whether chain elongation or initiation of the synthesis of replicating units is affected, and which step in either of these complex processes is defective. In order to facilitate the study of defects found in conditional mutants, we will continue our studies of the mechanism of DNA replication in the wild-type organism (both in vivo and in vitro) using the techniques of precursor incorporation, sucrose density gradient centrifugation, electron microscopy and fiber autoradiography. Comparison of the characteristics of wild-type strains and those of strains carrying ts dna genes should provide considerable information concerning DNA replication in this eukaryote. Eventually, the nature and function of the various specific proteins required for DNA replication will be investigated, using in vitro systems isolated from mutant strains in complementation assays for the purification of the proteins from extracts of the wild-type organisms. The elucidation of the mechanisms by which DNA replication is controlled in normal eukaryotes may lead to information as to its possible alteration in cancer cells, in which the regulation of cell division appears to be altered. Once such an alteration is identified, it may be subject to correction by selective adminstration of pharmacological or other agents. BIBLIOGRAPHIC REFERENCES: Nucleotide Metabolism in Physarum polycephalum: The Effect of Ionizing Radiation, H. H. Evans, S. R. Littman and T. E. Evans, Radiat. Res. 67, 531 (1976). Studies on Phleomycin Resistance in Physarum polycephalum, T. E. Evans, R. A. Biehler and H. H. Evans, 2nd Int. Mycological Congress, Tampa, Florida, 1977.